A method has been developed for the high-throughput inhibition screening of the major human cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) using an in vitro substrate cocktail and liquid chromatography-tandem mass spectrometry (LC-MS-MS). A cocktail consisting of the selective substrates phenacetin (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), bufuralol (CYP2D6), and midazolam (CYP3A4) was incubated with human liver microsomes. The metabolic reactions were terminated with methanol containing dextrorphan as an internal standard. Following centrifugation, the supernatant was analyzed by LC-MS-MS employing a fast gradient. The concentrations of the substrate metabolites-paracetamol, 4-hydroxytolbutamide, 5-hydroxyomeprazole, 1'-hydroxybufuralol, and 1'-hydroxymidazolam-in each sample were determined by LC-MS-MS in a single assay. The method was validated by incubating known CYP inhibitors (furafylline, CYP1A2; sulfaphenazole, CYP2C9; s-mephenytoin, CYP2C19; quinidine, CYP2D6; and troleandomycin, CYP3A4) with the individual substrates they were known to inhibit and with the substrate cocktail. IC50s (microM) determined using the substrate cocktail were in good agreement with those obtained with individual substrates (furafylline, 2.9 vs. 2.0; sulfaphenazole, 0.75 vs. 0.72; s-mephenytoin, 170 vs. 180; quinidine, 0.17 vs. 0.24; troleandomycin, 2.6 vs. 3.2) and with previously reported values in the literature.